Non-woven keratin cell scaffold

ABSTRACT

A hydratable, highly absorbent keratin solid fiber or powder capable of absorbing a large weight excess of water may be produced by partially oxidizing hair keratin disulfide bonds to sulfonic acid residues and reacting the sulfonic acid residues with a cation. The neutralized suspension can be filtered, washed, and dried, leaving keratin solid which can be shredded into fibers and further ground into powder. Addition of water to the solid produces a hydrogel. The powder or hydrogel may be useful as an absorbent material, as a therapeutic for skin, or as an excipient. The keratin materials can be incorporated into nonwoven films. The hydrogel may be used as biocompatible viscoelastic filler for implant applications. Both the hydrogel and nonwoven materials are also suitable for use as tissue engineering scaffolds.

I. RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 09/528,893, filed Mar. 20, 2000, now U.S. Pat. No.6,270,793, which is a continuation-in-part of U.S. application Ser. No.09/512918 filed Feb. 25, 2000, now U.S. Pat. No. 6,274,155, which is acontinuation-in-part of U.S. patent application Ser. No. 09/394,782filed on Sep. 13, 1999 now U.S. Pat. No. 6,316,598.

II. FIELD OF THE INVENTION

The present invention is related generally to a keratin composition andmethod for making same. More specifically, the present invention relatesto an absorbent keratin powder or fiber. In particular, the presentinvention includes a hydratable keratin solid which forms a hydrogelupon addition of water for use in various applications includingnonwoven films, diapers, skin treatments, prosthetic devices,excipients, tissue-engineering scaffolds and the like.

III. BACKGROUND OF THE INVENTION

Absorbent materials are currently used to absorb body fluids such asurine, menses, and wound exudate. The absorbent materials are placednear the skin to serve this purpose. One class of products includesdiapers, where the absorbent material can be derived from wood pulp,cellulosic fibers, or super absorbent, synthetically produced material.Diapers commonly have an inner core designed to absorb urine and water.The core is typically formed from a superabsorbent polymer dispersed ina larger amount of less absorbent material. The absorbent materialstypically contained in the core are separated from the skin by at leastone layer of material. The absorbent materials absorb urine and canbecome saturated. It is believed that some material from the absorbentcore leaches from the wet absorbent and travels back to the skin. In thecase of chemically treated absorbent materials and films, depending onthe chemicals, the leachate may be irritating and is not believed to bebeneficial. Skin contact with urine can also occur and result inirritation. This type of irritation may exacerbate diaper rash problems.

Other products which contain absorbent materials for use next to theskin include feminine hygiene products such as tampons and pads. Theseproducts serve to absorb menses. Another class of products usingabsorbent materials includes wound dressings, both those designed forhumans, and dressings for veterinary use for application to wounds orskin irritations or disorders in animals. For specific applications,wound dressings preferably absorb exudate from wounds while keeping thewounds relatively moist to promote healing. In some applications, a gelmay be desirable as a wound dressing, where the gel can maintain a moistwound environment, while absorbing excess exudate.

What would be desirable is an absorbent material formed from a naturalproduct. What would be beneficial is a non-toxic product derived fromnatural sources that would cause no concern when leachate from thematerial contacts the body or the material itself contacts the body.What would be advantageous is a material that can absorb urine and, whenwet, leach out a natural product that is beneficial with respect todiaper rash. What would be desirable is a material that can return askin healing leachate to the skin. What would be desirable is a materialthat aids wound healing. What would be desirable is a hydrogel made ofnatural products formable by adding water to a powder or fiber.

Tissue engineering is a rapidly growing field encompassing a number oftechnologies aimed at replacing or restoring tissue and organ function.The success of tissue-engineered implants rest on the invention ofbiocompatible materials that can act as cell-scaffolds and support cellgrowth. Of benefit are tissue-engineering scaffolds materials that aremitogenic or contain mitogenic factors. Such scaffolding materials canbe used for in a wide array of tissue engineering implants containingcellular components, such as, for example, osteoblasts, chondrocytes,keratinocytes, and hepatocytes, to restore or replace bone cartilage,skin and liver tissue respectively.

IV. SUMMARY OF THE INVENTION

The present disclosure addresses at least some of the deficiencies inthe art by providing a hydratable, hydrogel-forming solid derived from akeratinous source such as hair, fur, human hair and the like. In thecontext of the present invention, the term “hydratable keratin” and“hydratable keratin material” is a keratin or keratin material that whenhydrated can form a hydrogel. In certain embodiments, a hydrogel-formingsolid as disclosed herein may absorb up to 5 to 20 times its weight inwater to form a hydrogel. Such a solid, as well as the hydrogel formedfrom the solid will be useful in various applications such as use as anabsorbent with skin healing properties when incorporated into diapers,feminine hygiene products, wound dressings, including both human andveterinary uses, as a soft tissue augmentation medium when used insubdermal implants, tissue-engineering cell scaffolds, as a moisturecontaining agent in cosmetics, oils, lotions, or gels for use on theskin, in applications related to the healing of damaged skin, and as apharmaceutical excipient for sustained release pharmaceuticalapplications.

A hydratable keratin solid may be made by methods that include providinga keratinous material, or keratin, having disulfide linkages andpartially or substantially oxidizing the keratinous material with anoxidizing agent, for example, such that some disulfide linkages arecleaved and oxidized, forming hydrophilic sulfonic acid or cysteic acidresidues. A preferred source of keratinous material is human hair,although the keratin may be obtained from hair or fur of animalsincluding any mammal, or from finger or toenail material or from hooves,feet, beaks, skin, feather or horns. Human hair is a preferred source ofkeratin because of its ready availability from cuttings of barber andbeauty shops, because it is expected to be less prone to causeundesirable immune or allergic reactions in a human, and because akeratin preparation may be made from the hair of a subject for whom thepreparation will be used. This last advantage can be especiallyimportant in embodiments involving subdermal and tissue engineeringimplantations.

It is well known in the art that keratins contain substantial sulfur,that is, the amino acid sequence of keratin contains a high proportionof cysteine residues as compared to proteins in general. These cysteineseach include a sulfhydryl moiety that is able to bond with anothersulfhydryl moiety from another cysteine residue to form a disulfidebond. The second cysteine may reside within the same keratin molecule,or in another keratin molecule. These disulfide bonds are responsiblefor much of the tertiary and/or quaternary structure of this class ofproteins. A suitable oxidizing agent is able to break this disulfidebond and to oxidize one or both of the sulfhydryl moieties so that theyare no longer able to form a disulfide. Such an oxidation is a part ofthe process of forming the keratin products of the present disclosure.Preferred oxidizing agents include, but are not limited to peraceticacid, hydrogen peroxide, perborates, percarbonates, benzoyl peroxide, orammonium sulfate peroxide. However, any suitable oxidizing agent knownin the art can be used in the practice of the invention. Afteroxidation, the liquid oxidizing agent can be filtered from the oxidizedkeratin solid, and the solid may be washed to remove residual oxidizingagent, for example.

The resulting solid may then be suspended in a non-aqueous solvent andthe pH may be adjusted upward with base—conveniently to at least neutralpH. Preferred solvents for this second solution do not include more thanabout 20 volume percent water, as the water may hydrolyze the peptidebackbone during processing. Preferred solvents would include alcoholssuch as methanol, ethanol, or propanol, for example, and would alsoinclude non-aqueous polar, water-miscible solvents such as acetone andtetrahydrofuran, for example. An effective solvent should be able tosolvate a Lewis base and should also be able to provide a medium able tokeep the keratin sufficiently swelled to allow ionic associations orinteractions between the base cations and anionic sulfonic acid groupsin the keratin. Small amounts of water will assist in this regard, soblends of the aforementioned solvents in combination with water up to 20volume percent may be used. Preferred bases include, but are not limitedto sodium hydroxide, potassium hydroxide and ammonium hydroxide, which,as is known in the art, would yield or produce sodium, potassium andammonium cations, respectively, upon entering solution.

The keratin suspension may be heated, and is preferably heated toboiling for a time sufficient to swell the keratin. The keratinsuspension may be stirred without heat for a longer period of time toallow a more complete association or reaction between the sulfonic acidgroups and the base cations. The continued reaction time at or near roomtemperature, or even below room temperature while stirring iscontemplated by the inventors to allow the base cations to approach andbind to the keratin anionic sites with a lower incidence of peptidebackbone degradation that could occur with continued boiling. Thecations for use in the present invention, therefore, must be able tointeract with the anionic cysteic groups in the keratin material. Theuse of the term “cations” or “monovalent cations” in the presentdisclosure and claims is indication of those cations that are able to doso. After a sufficient reaction time, the keratin solid may be removedfrom the suspension by filtration, for example, and dried, leaving asolid salt formed of the keratin sulfonic acid or cysteic acid groupsand base cations. This solid may be shredded into a fibrous form and/orground into a finely divided powder. This solid may be used in certainembodiments, or it may be hydrated by adding water, for example, and thehydrogel, or viscoelastic hydrogel thus formed may be used in certainembodiments.

In certain embodiments, an absorbent keratin layer may be incorporatedinto various absorbent articles such as a disposable diaper, a wounddressing, or feminine hygiene product, by adsorbing or coating a keratinsolid or hydrogel onto a layer of the article, by impregnating acomponent of such an article, or by associating a keratin material witha nonwoven layer of such an article. In certain embodiments an absorbentkeratin powder may be applied directly to skin to absorb moisture andinhibit rashes or chafing, such as diaper rash, for example. Ahydratable keratin solid of the invention may have an absorbency of 1,5, 10, 15 or even up to 20 times its weight in water. The absorbency maybe adjusted by, for example, varying the degree of oxidation of thekeratin in the process. It may thus provide a substitute or a supplementfor products such as talc and cornstarch. The inventors havedemonstrated, for example, that a fibrous or powdered form of solidkeratin material as described herein can absorb about 10 times itsweight in water in about 10 seconds.

The hydratable keratin solids as described herein form a hydrogel or aviscoelastic hydrogel upon application of water, and also arecontemplated to contain skin healing peptides associated with thekeratin, which may leach out of the keratin products when wet. Thekeratin products thus provide an added benefit, in addition to waterabsorbency, that is, healing or soothing peptides are also released thatmay have beneficial effects on the skin of a user of the products. Thisproperty offers certain benefits in embodiments such as wound dressings,as well as cosmetics, gels or lotions for application to the skin.

In certain embodiments a keratin absorbent as disclosed herein may beused as a wound dressing material to absorb wound exudate by directapplication, or by incorporation into a dressing. The solid, hydratableforms of keratin offer certain advantages in such applications becausethey may be stored as dry powders or fibers and hydrated to form a gelin the field, or only as needed, for example. Medical applications, suchas wound exudate management or drug release, can make use of the keratinmaterial in absorbent powder, fiber, woven fiber, or felt form.

The keratin hydrogel is also believed to be suitable for use as animplant filler, for example, used to fill a breast implant, or toaugment soft tissue for cosmetic, reconstructive or aesthetic reasons,or in a tissue expander application. The keratin product may also beused in cosmetics to retain moisture next to the skin. The performanceof cosmetics which reduce the greasy appearance of skin can be enhancedthrough the use of moisture absorbent keratin material as an additive orbase ingredient, for example, in a cosmetic formulation. The keratinabsorbent and hydrogel can also be used for a variety of tissueengineering applications. Both materials may act as biocompatiblescaffolds that provide a mitogen, the keratin peptide, to the cellularcomponents of a tissue-engineered implant.

The present invention may be described, therefore, in certain aspects asa composition comprising a hydratable keratin solid, wherein the solidcomprises a keratin where at least a portion of the cysteic groups ofthe keratin are ionically or electrostatically associated with, or maybe ionically bound to cations. As used herein, ionically bound orionically associated would have their ordinary meaning as is known inthe art, and would include the electrostatic attraction between an anionand a cation, and would include such interactions directly, such asthrough formation of ionic bonds, and interactions through intermediarybipolar moieties, for example. A cysteic group would include cysteineand derivatives of cysteine including cysteine and cysteic acid orsulfonic acid. As used herein, cysteic acid and sulfonic acid denote acysteine side chain in which the terminal sulfur is bonded to threeoxygen atoms to produce the sulfonic acid ion, SO₃ ⁻, or the acidicform, SO₃H. In certain embodiments, a portion of the cysteic groups areoxidized to sulfonic acid or cysteic acid groups. Sulfonic acid orcysteic acid groups may comprise a significant portion of the totalcysteic groups and in some embodiments the sulfonic acid groups mayconstitute a major portion of the total cysteic groups. The extent ofthe oxidation may be adjusted by adjusting certain parameters of theoxidation reactions, such as temperature, concentration of oxidizingagent, and time of reaction, for example, to achieve a product withcertain desired properties, such as absorbency or resiliency, forexample.

In certain embodiments, therefore, the present invention may bedescribed as a hydratable keratin solid made by a process comprisingoxidizing a portion of the cysteic acid groups of a keratin to obtain akeratin having oxidized cysteic groups, and contacting the keratinhaving oxidized cysteic groups with monovalent cations under conditionseffective to form ionic associations or ionic bonds between at least aportion of the oxidized cysteic groups and the cations.

In some embodiments, the hydratable keratin solid is made by a processcomprising oxidizing at least a portion of the cysteic acid groups of akeratin to obtain a keratin having oxidized cysteic groups, andcontacting said keratin having oxidized cysteic groups with monovalentcations under conditions effective to form ionic associations or ionicbonds between a substantial portion of said oxidized cysteic groups andsaid cations. The oxidation may comprise placing the keratin in asolution containing a concentration of an oxidizing agent effective tooxidize a portion of the cysteic acid groups. The portion of oxidizedcysteic groups may be a major portion of the total cysteic acid groups.

In certain embodiments of the present invention, the oxidation comprisesplacing the keratin in a solution containing a concentration of hydrogenperoxide, peracetic acid, perborates, percarbonates, benzoyl peroxide orammonium sulfate peroxide effective to oxidize a portion of the cysteicgroups.

The process of the present invention may further comprise heating thekeratin solid containing oxidized cysteic groups in a solvent solutioncontaining a dissolved base, wherein the base produces the monovalentcations in the solution. The solvent solution may comprise a solventselected from methanol, ethanol, propanol, ether, tetrahydrofuran (THF),acetone, propylene glycol, 1,4-dioxane, and glycol ether, orcombinations of these with up to 20 volume percent water. In certainembodiments the process further comprises removing the solution from theheat and stirring for a time effective to form ionic bonds between thecysteic groups and cations produced by the base. The process may alsofurther comprise drying the keratin solid, such as by drying a solid orsolution under vacuum.

Another aspect of the present invention is a composition comprising akeratin hydrogel wherein the hydrogel is produced by adding water to acomposition comprising a hydratable keratin solid, wherein the solidcomprises a keratin where at least a portion of the cysteic groups ofthe keratin are ionically bound to cations. In some embodiments, thecomposition of the present invention comprises a keratin viscoelastichydrogel produced by adding water to a composition comprising ahydratable keratin solid, wherein the solid comprises a keratin where aportion of the cysteic groups of the keratin are ionically bound to orassociated with cations.

Another aspect of the present invention is a process for making ahydratable keratin solid comprising: (1) oxidizing keratin in a firstsolution comprising a soluble oxidizing agent, such that a portion ofthe disulfide bonds of the keratin are oxidized to form sulfonic acidresidues, to obtain an oxidized solid fraction; (2) separating theoxidized solid fraction from the first solution; (3) contacting theoxidized solid fraction with a second, basic solution comprising amonovalent cation dissolved in a solvent or solvent mixture; (4)maintaining the second solution containing the oxidized solid fractionand the monovalent cations for a time and at a temperature effective toallow an interaction between the sulfonic acid residues and themonovalent cations to obtain a salt solution of the keratin and themonovalent cation; and (5) substantially removing the solvent from thesalt solution to obtain a pure hydratable keratin solid.

The process may also further comprise adjusting the pH of the secondsolution, to obtain a substantially neutral solution. In someembodiments, the keratin is obtained from hair, fur, skin, feet, beaks,horns, hooves or feathers and is preferably obtained from human hair.

In some embodiments, the keratin is oxidized by suspending the keratinin a solution of a suitable oxidizing agent such as one selected fromthe group consisting of hydrogen peroxide, peracetic acid, perborates,percarbonates, benzoyl peroxide, and ammonium sulfate peroxide, in aconcentration of between about 1 and about 35 weight/volume percent. Invarious embodiments, the keratin is oxidized by suspending the keratinin a solution of an oxidizing agent selected from the group consistingof hydrogen peroxide, peracetic acid, perborates, percarbonates, benzoylperoxide, and ammonium sulfate peroxide, in a concentration of about 1,or about 2, or about 3, or about 4, or about 10, or about 15, or about20, or about 30, or about 32, or about 35 weight/volume percent. As usedherein the term weight/volume percent refers to a solution in which theconcentration is determined in weight percent, that is then diluted intoa particular volume, arriving at a weight/volume percent. For example,in order to arrive at the oxidant solutions described herein a “stocksolution” at fairly high concentration is diluted in water. As anexample, hydrogen peroxide may be purchased as a 30 weight % solution(30 grams of peroxide per 100 grams of solution). To make 1 liter of a2% solution of this, one would dilute 66.7 mL of the 30 weight % stocksolution in 933.3 mL of water. The net effect is to cut the stocksolution 15-fold (from 30 down to 2%). This ratio is a weight to volumeratio, so the resulting solution is described as 2 weight/volume %.

In some embodiments, the keratin is oxidized by suspending the keratinin a solution of a suitable oxidizing agent, such as one selected fromthe group consisting of hydrogen peroxide, peracetic acid, perborates,percarbonates, benzoyl peroxide, and ammonium sulfate peroxide, in aconcentration of between about 1 and about 35 weight/volume percent, ata temperature between about 0° C. and about 100° C. In other embodimentsthe temperature is between about 4° C. and about 90° C., or betweenabout 20° C. and about 100° C., or between about 80° C. and about 100°C.In other embodiments, the temperature is about 4° C., or about 90° C.,or about 100° C.

The present invention may also include the process wherein the keratinis oxidized by suspending said keratin in a solution of an oxidizingagent selected from the group consisting of hydrogen peroxide, peraceticacid, perborates, percarbonates, benzoyl peroxide, and ammonium sulfateperoxide, in a concentration of between about 1 and about 35weight/volume percent, at a temperature between about 0° C. and about100° C. for a period of between 0.5 and about 24 hours, or in aconcentration of oxidizing agent of between about 1 and about 35weight/volume percent, at a temperature between about 0° C. and about100° C. for a period of between 1 and about 2 hours, or for betweenabout 2 and about 4 hours, or for between about 1 and about 4 hours, orfor a period of about 10 hours.

More specifically, the present invention may include oxidizing thekeratin by suspending the keratin in a solution of between about 1percent to about 32 percent peracetic acid at a temperature betweenabout 0° C. and about 100° C. for between about 0.5 and about 24 hours,or by suspending the keratin in a solution of about 1 percent peraceticacid at a temperature between about 1° C. and about 100° C. for betweenabout 0.5 and about 24 hours, or by suspending the keratin in a solutionof between about 4 percent peracetic acid at a temperature of about 4°C. for 24 hours, or by suspending the keratin in a solution of about 4percent peracetic acid at room temperature for about 24 hours, or bysuspending the keratin in a solution of about 4 percent peracetic acidat about 90° C. for about 10 hours, or by suspending the keratin in asolution of about 4 percent peracetic acid at a temperature betweenabout 20° C. and about 100° C. for between about 1 and about 4 hours, orby suspending the keratin in a solution of about 4 percent peraceticacid at a temperature between about 80° C. and about 100° C. for betweenabout 1 and about 2 hours, or even by suspending the keratin in asolution of about 2 percent peracetic acid at a temperature betweenabout 1° C. and about 100° C. for about 2 hours.

A second solution in the process of making the disclosed compositions,wherein the second solution contains the oxidized solid fraction andmonovalent cations may be heated, and may also be boiled for betweenabout 0.5 hours and about 12 hours, for between about 0.5 hours andabout 3 hours, or for about 1 hour. Once said solution is boiled, thesolution may be allowed to continue reacting while being stirred afterremoval of the heat. Alternatively, the solution may be stirred andallowed to react without the application of heat, or of boilingtemperatures. In certain embodiments, the solution is allowed to reactat a temperature of between about 15° C. and about 30° C. for a periodof between about 1 and about 24 hours, or at a temperature of betweenabout 20° C. and about 25° C. for a period of between about 1 and about5 hours, or at room temperature for a period of about 5 hours. Incertain preferred embodiments the solution is heated to the boilingpoint of the solvent and boiled for 2 hours.

Certain processes as described herein are effective to produce ahydratable keratin solid, and it is an embodiment of the presentinvention that those solids may be hydrated by the addition of water toobtain keratin hydrogels, or even viscoelastic keratin hydrogels. Theterms hydrogel and viscoelastic hydrogel, as used herein, are meant tohave the art recognized definition, and could be described as absorbingwater such that the water cannot be removed by mechanical methods suchas pressure or centrifugation. Viscoelastic hydrogels would also bedefined as gels that display non-Newtonian fluid properties.

In certain embodiments the present invention may be described as adisposable diaper that includes a hydratable keratin solid, or a diaperwhich incorporates a hydratable or absorbent keratin solid. A hydratablekeratin solid may be coated on a layer of the diaper, either a layernext to the skin of a wearer, or a layer separated from the skin of awearer by a water permeable layer. In certain embodiments a hydratablekeratin solid may be associated with a nonwoven layer of a diaper, ormay be impregnated into a layer of a disposable diaper, or it may becontained in an inner absorbent core.

In certain alternative embodiments, the present invention may bedescribed as a feminine hygiene product, or a wound dressing thatincludes a hydratable keratin solid. As was described for use indiapers, a hydratable keratin may be coated on a layer of a product,associated with a nonwoven layer of a product, or even impregnated intoa layer of a product or contained in an absorbent core. Exemplaryproducts would include wound dressings, tampons, and sanitary pads.Wound dressings include absorbent wound dressing, that is dressingcapable of, but not limited to, absorbing wound exudate and blood.Absorbent dressings include, but are not limited to, adhesive bandages.Adhesive bandages typically comprise an absorbent pad, a backing and apressure sensitive adhesive to maintain the dressing in place. In oneaspect of the present invention, an absorbent wound dressing comprisinghydratable keratin is an absorbent pad of an adhesive bandage. In oneembodiment, the hydratable keratin in such an absorbent pad is in anonwoven film.

Certain embodiments of the invention may be described as methods forpromoting healing of skin in a subject including a human or an animalhaving damaged skin, including providing an absorbent, keratin material,wherein a portion and preferably a substantial or major portion of thecysteic groups of said keratin are oxidized and wherein water solublepeptides are associated with the keratin, wherein at least some of saidpeptides can leach out from said keratin upon application of water, andwherein said peptides promote healing of damaged skin; and disposing theabsorbent keratin material near damaged skin, such that moisture causesat least some of said peptides to leach out of said keratin and tocontact said skin. The method may be practiced with animal or humansubjects, such that either animal or human skin is healed by thismethod. The practice of the method for promoting skin healing asdescribed herein may include the treatment of damaged skin including,but not limited to diaper rash, burn, sunburn, cut, abrasion, puncture,a sore, bed sore, ulcer, diabetic ulcer, irritated skin, surgicalincision, skin graft donor site, or wrinkled skin. The keratin materialmay be incorporated in a nonwoven film. The nonwoven film may comprisesynthetic polymer webs and may also comprise natural materials such ascotton. It is understood that in the practice of such embodiments, thewound of the subject being treated may exude or excrete moisture andthat the absorption of such moisture by said keratin may cause therelease of water soluble peptides from keratin products of the presentinvention.

In certain embodiments the present invention may be described as amethod for promoting skin healing, in particular in those embodiments inwhich a keratin solid or hydrogel as described herein, such as a keratinsolid or hydrogel in which the keratin is obtained from human hair, forexample, is contained in, or forms a portion of a cream, lotion, or gelfor application to skin, hair, lips, or nails, for example. Suchformulations can offer various advantages such as moisturizing the skin,or inhibiting loss of moisture from the skin, as well as providing thehealing effects of peptides that may leach from the keratin containingproduct. Such creams, lotions and gels may be applied to damaged skin,such as dry, burned, sunburned, wrinkled, cut, scraped, chapped,irritated, ulcerated or otherwise damaged skin or other tissue.

One aspect of the present invention is a nonwoven film compositioncomprising a synthetic polymer and a keratin material, wherein thekeratin material has been oxidized and contains sulfonic acid groups.The keratin material may be oxidized keratin material that has notundergone the ion exchange process or may be oxidized keratin that hasundergone the ion exchange process to form a hydratable keratin. In thelatter, the sulfonic acid groups of the hydratable keratin areassociated with monovalent cations. The keratin materials may also beassociated with pharmaceutical agents which may be in the form of polarcompounds which are capable of binding or otherwise associating with thekeratin. Such a pharmaceutical agent is aspirin. The synthetic polymermay be, but is not limited to, α-olefins, acrylates, urethanes,acetates, nylons, esters, and copolymers thereof. An α-olefin isconsidered to be any monomer containing an α-double bond. The nonwovencomposition may also further comprise a natural material which may be,but is not limited to, cotton. In some embodiments of the invention, thenonwoven composition is a laminate, which may be, but is not limited to,a tri-laminate comprising two outer layers of synthetic polymer and amiddle layer of keratin material. The keratin material in the middlelayer may be partially exposed by openings in the two outer nonwovensynthetic polymer layers. In some embodiments of the invention thesynthetic polymer layers are nonwoven webs of polymer fibers. In otherembodiments, the synthetic polymer layers are woven webs of polymerfibers.

Another aspect of the invention is a nonwoven tri-laminate compositioncomprising a middle layer of a keratin material between two outer layersof synthetic polymer material. The synthetic polymer may be in the formof a nonwoven web. The keratin material may be oxidized keratin thatcontains sulfonic acid residues. The oxidized keratin may be subjectedto ion exchange such that the keratin material is a hydratable keratinmaterial. The keratin material may be associated with pharmaceuticalagents, which may be in a cationic form. The synthetic polymer may be,but is not limited to, α-olefins, acrylates, urethanes, acetates,nylons, esters, and copolymers thereof. Ester polymers include, but arenot limited to poly-(α-hydroxy acids) such as polylactic acid andpolyglycolic acid and their co-polymers. The nonwoven composition mayalso further comprise a natural material which may be, but is notlimited to cotton.

One aspect of the invention is a process for making a nonwoven film. Inone embodiment a keratin material is applied to a first nonwoven weblayer of synthetic polymer. A second nonwoven web layer of syntheticpolymer is applied over the keratin material so as to form atri-laminate composition with two outer layers of nonwoven syntheticpolymer web and a middle layer of keratin material. Another aspect ofthe invention is a product made by the above described process. Thekeratin material may be oxidized keratin that contains sulfonic acidresidues. The oxidized keratin may be subjected to ion exchange suchthat the keratin material is a hydratable keratin material. The keratinmaterial may be associated with pharmaceutical agents, which may be in acationic form. The synthetic polymer may be, but is not limited to,α-olefins, acrylates, urethanes, acetates, nylons, esters, andcopolymers thereof The nonwoven composition may also further comprise anatural material which may be cotton. The keratin material in the middlelayer may be partially exposed by openings in the two outer nonwovensynthetic polymer layers.

Other aspects of the present invention include wound dressings, diapersand feminine hygiene products which comprise a nonwoven film made from asynthetic polymer and a hydratable keratin material. In certainembodiments, the non-woven film of the present invention may be next tothe skin or other epithelial layer of a subject, or may be separatedfrom the skin or other epithelial layer of a subject by a waterpermeable layer, which may be a non-wetting water permeable layer. Incertain embodiments a hydratable keratin solid may be associated with anonwoven layer of a diaper, or may be impregnated into a layer of adisposable diaper, or it may be contained in an inner absorbent core.These products may be laminate compositions, which may be tri-laminatescomprising two outer layers of synthetic polymer and a middle layer ofkeratin material. The keratin material in the middle layer may bepartially exposed by openings in the two outer nonwoven syntheticpolymer layers. In some embodiments of the invention the syntheticpolymer layers are nonwoven webs of polymer.

Creams, lotions, or gels of the present invention may incorporate orreplace other ingredients known in the art, including, but not limitedto oleaginous, emulsifiable, emulsion base, or water-soluble ointmentbases as are well known in the pharmaceutical arts. Oleaginous basesthat may be combined with the keratin compositions include ointmentscontaining white wax and/or white petrolatum, ointments containingyellow wax and petrolatum, cetyl esters wax, oleic acids, and paraffins.Absorbent ointment bases or emulsifiable bases that may be used includethose containing anhydrous lanolin, or combinations of cholesterol,stearyl alcohol, white wax and petrolatum, for example. Emulsion basesand components that may be used include ointments containing cetylalcohol, and cold creams such as those containing cetyl esters wax,white wax, mineral oil, sodium borate and water, for example. Otherointments of the present invention may contain glyceryl monostearate,lanolin, stearic acid, or a combination of methylparaben, propylparaben,sodium lauryl sulfate, propylene glycol, stearyl alcohol and whitepetrolatum, for example, or an ointment containing cetyl esters wax,white wax, almond oil, sodium borate, stronger rose water, and rose oil,for example. Water soluble ointments and creams for use in the presentinvention may include glycol ethers and derivatives thereof,polyethylene glycols, polyoxyl 40 stearate, and/or polysorbates.

The preparations as described herein for topical applications may alsoinclude protectives and absorbents, demulcents such as benzoin,hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylalcohol, propylene glycols, sodium alginates, and tragacanth.Emollients, astringents, or antiperspirants may also be included in thekeratin containing formulations as described herein.

An aspect of the present disclosure is a method for augmenting softtissue in a subject comprising injecting a keratin composition asdescribed herein subdermally in an area in need of augmentation. Avariety of such applications are available in light of the presentdisclosure and would include augmentation of soft tissue including, butnot limited to bulking of a urinary sphincter in order to alleviateurinary incontinence, augmentation of vocal chords to restoreelasticity, as well as improvement of the appearance of a subject byaugmentation of breasts, lips, chin, gluteal area, or even to improvewrinkled or acne scarred skin, or skin scarred by other conditions, andincluding soft tissue voids or indentations. A keratin composition maybe provided as a dry solid and hydrated after subdermal implantation, ora hydrogel or viscoelastic hydrogel may be prepared and implanted. Incertain embodiments, a dry or hydrated keratin material may be containedin a biocompatible envelope, bag, or container for subdermalimplantation, and hydrated after implantation by addition of water orabsorption of body fluids, or a keratin material may be suspended in aninjectable carrier and injected in the desired area of augmentation. Inone embodiment, soft tissue augmentation is accomplished by injecting apreparation comprising a keratin hydrogel and a cell population.

Another aspect of the present invention is the use of keratincompositions for tissue engineering applications. One embodiment is animplantable preparation comprising a keratin hydrogel and a cellpopulation. The cell population may include, but are not limited to,keratinocytes, fibroblasts, chondrocytes, hepatocytes, splenocytes,neurocytes, osteoblasts, or endothelial cells. The keratin hydrogelpreparation may be prepared such that it is implantable by injection.The keratin hydrogel may also contain a therapeutic agent, which may bea water soluble peptide, which may be a mitogen.

Another embodiment is a cell scaffold comprising a nonwoven film whichcomprises a hydratable keratin which contains sulfonate groups. Thenonwoven film may comprise a synthetic polymer which may be resorbable.The nonwoven film may also be a laminate composition. The hydratablekeratin may contain a therapeutic agent which may be a water solublepeptide, which may be a mitogen.

Another embodiment of the present invention is a method of implanting apreparation comprising a population of cells and a keratin hydrogel intoan animal. The cell populations may include, but are not limited to,keratinocytes, fibroblasts, chondrocytes, hepatocytes, splenocytes,neurocytes, osteoblasts, or endothelial cells. The hydrogel may containa therapeutic agent which may be a water soluble peptide, which may be amitogen. A further embodiment is a method of implanting a cell scaffoldcomprising a nonwoven film that comprises a hydratable keratin whichcontains sulfonate groups. Cells may be grown on the nonwoven cellscaffold in vitro. Thereby, the nonwoven cell scaffold may be seededwith cells prior to implantation. Such cells include, but are notlimited to, keratinocytes, fibroblasts, chondrocytes, hepatocytes,splenocytes, neurocytes, osteoblasts, or endothelial cells. The nonwovencell scaffold may be used to repair damaged hard and soft tissues. Hardtissues include, but are not limited to, bone and cartilage. Softtissues include, but are not limited to, skin, mucosa and muscle.Mucosal tissue includes, but is not limited to, gingival tissue. whichmay be damaged bone or damaged cartilage. Another embodiment comprisesimplanting a keratin hydrogel cell scaffold. A therapeutic agent may beincluded in the keratin component of all the cell scaffolds of thepresent invention. Such a therapeutic agent may leach out of the keratinand may be a water soluble peptide, which may be mitogenic.

It is an aspect of the present invention that a keratin composition asdescribed herein, and in particular keratin obtained from human hair isalso useful as an excipient for the delivery of a pharmaceutical agent,and in particular in the sustained or delayed release of apharmaceutical agent. An embodiment of the invention may be described,therefore, as a composition comprising a keratin having oxidized cysteicgroups and a pharmaceutically active agent. Such a formulation mayinclude a hydratable keratin solid excipient, or a keratin hydrogeldepending on the particular application.

In the practice of the invention, a dry hydratable keratin as describedherein may be mixed with a powdered pharmaceutical agent and water addedto hydrate the mixture, or alternatively such a solid mixture may beformulated as a compressed tablet to be orally administered or forextemporaneous preparations for injection, or as a molded tablet, or itmay be enclosed in a capsule for oral administration or subdermalimplantation, for example. In certain embodiments a solution containinga water soluble drug or pharmaceutical agent may be added to ahydratable keratin so that the agent is carried into a hydrogel alongwith the water. A prepared hydrogel, or dry formulation may also beenclosed in a digestible or biodegradable capsule, such as a hardgelatin capsule for oral administration. In certain embodiments, thedescribed pharmaceutical preparations may be formulated for injection,either intravenous, subcutaneous, or intramuscular, for example, or forinhalant, for eye, ear, or nose drops, or for administration as asuppository.

In certain embodiments an active pharmaceutical agent may be associatedwith a keratin excipient by non-covalent attraction or association,through electrostatic, hydrophobic or ionic interaction, for example, orit may be covalently attached to a keratin excipient by covalent bondingto an oxidized keratin as described herein. In certain embodiments anactive agent such as a drug is physically or sterically entrapped withina keratin hydrogel and released over time by diffusion, or as a keratinexcipient is degraded.

It is understood that the pills formulated for oral administration,including a hydratable keratin solid, or even pills, capsules or tabletscontaining a keratin hydrogel may contain ingredients to serve ascoatings, additional fillers, binders and for color coding purposes.These ingredients are in common use in present pharmaceuticalformulations and may include, but are not limited to, gelatin, lactose,corn starch, calcium phosphate, povidone, magnesium stearate, stearicacid, colloidal silicon dioxide, hydroxypropyl methylcellulose,polyethylene glycol and one or more of the following dyes: FD&C Blue No.I Lake, FD&C Blue No.2 Aluminum Lake, D&C Green No. 5, D&C Yellow No.10, FD&C Yellow No. 6 or FD&C Red No. 3. Of course these are onlyexemplary fillers and dyes, those of skill in the art will recognizethat other inactive ingredients may be used in the preparation of theformulations of the present invention.

Keratin excipient preparations as described herein may be prepared fororal administration, and would also include injectable solutions orsuspensions for intramuscular or subcutaneous implantation includinglong acting injections, suppositories, topical ointments and transdermalapplications such as skin patches. Other ingredients may include asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride.

Suitable pharmaceutical agents for use with the excipients describedherein would include any pharmaceutical agent that can form anassociation with the keratin formulations through non-covalent,covalent, or steric; interaction. These agents would include proteintherapeutic agents, such as growth factors. In regard to oraladministration, such agents may include compounds such as such asacetaminophen, tetracyclines, penicillins, vitamins, antacids,non-steroidal antiinflammatory agents, anesthetics, breath fresheners,and minerals, for example.

In those embodiments in which transdermal administration is desired, thedisclosed compositions may be formulated to be administered by use of askin patch, or transdermal delivery system. Transdermal administrationmay be accomplished by any of a number of systems known in the art.Examples of systems that may be adapted for use with the compositionsdescribed herein include those systems of transdermal administrationdescribed in U.S. Pat. Nos. 4,816,252; 5,122,382; 5,198,223; 5,023,084;4,906,169; 5,145,682; 4,624,665; 4,687,481; 4,834,978; and 4,810,499(all incorporated herein by reference.)

These methods typically include an adhesive matrix or drug reservoirsystem and may include a skin permeation enhancement agent such asethanol, polyethylene glycol 200 dilaurate, isopropyl myristate,glycerol trioleate, linolenic acid saturated ethanol, glycerolmonooleate, glycerol monolaurate, n-decyl alcohol, capric acid, andcertain saturated and unsaturated fatty acids, and their esters,alcohols, monoglycerides, acetate, diethanolamides andN,N-dimethylamides (See for examples, U.S. Pat. No. 4,906,169).

The release rate of a keratin excipient preparation is determined by therate at which water is absorbed and the keratin solid disintegrates. Thewater absorption rate of the solid keratin can be controlled by thenumber of sulfonic acid residues generated in the oxidation procedure.By exposing the keratin source material to extremes of oxidantconcentration, temperature, and time, extremes of absorption rate can beobtained. For example, at low oxidant concentration, colder temperaturesand short time periods, relatively few disulfide residues will beconverted to sulfonic acid residues. Such a keratin solid, furtherprocessed as described herein will absorb relatively little water anddisintegrate relatively slowly. Conversely, a keratin solid prepared athigh oxidant concentration, at boiling temperature for a long timeperiod, further processed as described herein, will absorb relativelylarge amounts of water and disintegrate relatively quickly.Disintegration rates between these extremes can be obtained byprocessing the keratin source material using intermediate conditions.

In some embodiments of the invention, a composition for the delivery ofpharmaceutical agents is in the form of a nonwoven film comprising asynthetic polymer and a keratin material. The synthetic polymer may be,but is not limited to, α-olefins, acrylates, urethanes, acetates,nylons, esters, and copolymers thereof. In some embodiments the nonwovencomposition is a laminate, which may be a tri-laminate comprising twoouter layers of synthetic polymer and a middle layer of keratinmaterial. The keratin material in the middle layer may be partiallyexposed by openings in the two outer nonwoven synthetic polymer layers.In some embodiments of the invention the synthetic polymer layers arenonwoven synthetic polymer webs. The nonwoven film pharmaceuticaldelivery composition may be used externally or internally.

V. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments herein.

FIG. 1. Release of albumin from keratin tablets.

FIG. 2. Schematic of proposed mechanism for amine drug binding tooxidized keratin.

FIG. 3. Hydrogen peroxide oxidized hair pH 7 titration curve.

FIG. 4. Release of norephedrine from keratin tablets in simulatedgastric fluid.

FIG. 5. Release of acetaminophen from keratin tablets in simulatedgastric fluid.

FIG. 6. Loading of acetaminophen into keratin tablets. Loading based onamount used in ion exchange process.

FIG. 7. Stability of keratin hydrogel prepared by peracetic acidoxidation and at a solid to liquid weight ration of 1:8. Columns of <1day did not form a gel.

FIG. 8. Stability of keratin hydrogel prepared by peracetic acidoxidation and at a solid to liquid weight ratio of 1:6. Columns of <1day did not form a gel. Columns with data labels indicate gel stable atlast observation point.

FIG. 9. Stability of keratin hydrogel prepared by peracetic acidoxidation and at a solid to liquid weight ratio of 1:4. Columns of <1day did not form a gel. Columns with data labels indicate gel stable atlast observation point.

FIG. 10. Stability of keratin hydrogel prepared by hydrogen peroxideoxidation and at a solid to liquid weight ratio of 1:8. Columns of <1day did not form a gel.

FIG. 11. Stability of keratin hydrogel prepared by hydrogen peroxideoxidation and at a solid to liquid weight ratio of 1:4. Columns of <1day did not form a gel. Columns with data labels indicate gel stable atlast observation point.

FIG. 12. Stability of keratin hydrogel prepared by hydrogen peroxideoxidation and at a solid to liquid weight ratio of 1:4. Columns of <1day did not form a gel. Columns with data labels indicate gel stable atlast observation point.

VI. DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a hydratable solid derived from keratinwhich is highly absorbent and can form a hydrogel or viscoelastichydrogel upon the application of water. The keratin solid can includeprotein having an ionizable pendant group such as sulfonic acid whichcan be derived from an oxidized protein disulfide linkage. A preferredsource of protein is keratin, and particularly preferred is keratinobtained from hair, including human hair. While hair is a preferredsource of keratinous material, other keratinous materials are alsobelieved suitable for use in the present invention. Examples of othersources include animal hair, skin, hooves, feathers, beaks, feet andhorns. The patient or a human donor are some preferred sources of hair,as hair from these sources is most likely to result in a non-immunogenicproduct, although animal hair may be acceptable for many individuals formany applications. In one method according to the present invention,hair is provided, preferably clean and unbleached. In another method,the hair is washed with Versa-Clean™ (Fisher Scientific, Pittsburgh,Pa.), rinsed with deionized water, and allowed to dry.

A. Preparation of Oxidized Keratin

The hair can be oxidized in peracetic acid or another suitable reagentsuch as H₂O₂. One method utilizes between about 1% to 32% peraceticacid, at a temperature between about 0 degrees C. and 100 degrees C. forbetween 0.5 and 24 hours. In one method, about 1 weight/volume percentperacetic acid is used. One method treats 30 grams of hair with 500 mLof 4% peracetic acid at 4 degrees C. for 24 hours. Another method treatsthe hair at room temperature for 24 hours. Yet another method treats thehair at about 90 degrees C. for about 10 hours. In a preferred method,the hair is treated by heating the hair in the oxidizing agent forbetween about 1 and 4 hours at a temperature between about 20 and 100degrees C. In a more preferred method, the hair is treated by heatingthe hair in the oxidizing agent for between about 1 and 2 hours at atemperature between about 80 and 100 degrees C. In a most preferredmethod, the hair is treated by heating the hair in about 2 weight/volumepercent oxidizing agent for about 2 hours at a temperature of about 100degrees C. The oxidation is believed to cleave a significant portion ofkeratin disulfide bonds forming cysteic acid residues having sulfonicacid groups. The sulfonic acid groups are believed to be hydrophilic innature and will ionically bond to cations later in the process, forminga salt of the keratin and cation. The partial oxidation is also believedby Applicants to form or release short chain peptides, which can remainassociated with, or entrained in the keratin structure.

After oxidation, the keratin solid can be recovered from the oxidizingliquid using filtration or other suitable methods such as centrifugationor decantation. The recovered, oxidized solid can be washed with wateror alcohol such as methanol or ethanol to remove the excess oxidizingagent. In a preferred embodiment, washing is limited to avoid removingtoo much of any soluble peptide chains entrained in the keratin.

B. Preparation of Hydratable Keratin

The solid fraction can be suspended in a suitable solvent or solventmixture. The solvent should be capable of at least suspending the hairor keratin solid and keeping the solid sufficiently swelled forsubsequent reaction. The solvent is preferably a non-aqueous solvent, asthe presence of water can act to hydrolyze peptide backbone bonds, whichcan result in an inferior product. The solvent should be able tosolubilize the later added base. One group of suitable solvents includesalcohols such as methanol and ethanol. Other solvents such as ether,tetrahydrofuran (THF), acetone, propylene glycol, 1,4-dioxane and glycolethers may also be suitable as solvents. Small amounts of water willassist in swelling the keratin and may therefore be added to theaforementioned solvents in an amount up to 20 volume percent. Thesolvent used is preferably volatile to promote evaporation from thefinal solid product.

The hair or keratin solvent suspension can then have the pH titratedupward to at least about pH 7. Increasing the pH deprotonates thesulfonic acid groups, leaving the sulfonic acids free to exchange withanother cation. The pH can be adjusted with a base, preferably having amonovalent cation. Preferred bases include sodium hydroxide andpotassium hydroxide.

The pH-adjusted keratin suspension can be heated for a time andtemperature sufficient to swell the keratin structure and promoteneutralizing of the sulfonic acid sites with the provided cation. In apreferred method, the keratin suspension is boiled between about 0.5hours and 12 hours. More preferably, the keratin suspension is boiledbetween about 0.5 hours and 3 hours. In one method, the keratinsuspension is boiled for about 1 hour. Boiling for too long a timeperiod leads to a mushy keratin which results from degradation of thepeptide backbone. A hydrated keratin product is less preferred due tothe greater difficulty of grinding the keratin.

After boiling, the keratin is preferably allowed to continue to reactwith the provided base cation at lower temperature and with stirring.The lower temperature reaction preferably takes place at a temperatureof between about 15 and 30 degrees C. for between about 1 and 24 hours.More preferably, the lower temperature reaction takes place at atemperature of between about 20 and 25 degrees C. for between about 1and 5 hours. In one method, the keratin suspension is allowed to reactwith stirring at room temperature for about 5 hours. In certainembodiments the reaction is held at the boiling point of the solvent forabout 2 hours.

After reacting at lower temperature, the reacted solid can be separatedfrom the solvent using any suitable method such as filtration. The solidis preferably washed with a solvent such as the same solvent used in thereaction. Washing the keratin removes some of the base, which ispreferably removed. The base is preferably removed to make the keratinsolid less caustic.

After filtration and washing, the keratin can be dried by a method suchas evaporation under vacuum. In one method, the keratin is dried at roomtemperature under about 5 mm Hg vacuum for about 2 hours. The driedkeratin is preferably somewhat brittle, which can result in a betterproduct after grinding. The dried keratin can be shredded into fibersand can further be ground into a powder. The dried keratin can bedirectly ground into a powder using a mortar and pestle, a ball mill, orother means of breaking down or comminuting the dried keratin intoparticles. Alternatively, the keratin can be ground or milled in thesolvent used for said neutralization step.

One resulting hydratable fiber or powder has been observed to absorbabout 10 to 13 times its own weight in water. In one test, fibers havinga length of between one quarter and one-half inch were observed toabsorb an average of 1300% +/−33% of their weight in water at atemperature of 21.5 degrees C. The fiber has been observed to absorb atleast 10 times its own weight in water within about 10 seconds. Thepowder has been observed to rapidly absorb water as well.

The fibers were also tested for various toxicity parameters and werefound to be non-toxic, non-irritating, non-sensitizing as indicated inTable 1. In addition, the material is non-mutagenic as measured by theAmes test.

TABLE 1 Hydratable Keratin Toxicity Testing Data Test Standard ProtocolResult Acute Oral Toxicity Biological Evaluation of Medical Devices-Non-toxic at 2 g/kg Part 11: Tests for Systemic Toxicity; ISO 10993-11,1993. Acute Dermal Toxicity Biological Testing of Medical and DentalNon-toxic at 2 g/kg Materials and Devices - Part 10: irritation andSensitization Tests; ISO 10993-10, 1995. Acute Irritation BiologicalTesting of Medical and Dental Non-irritating at Materials and Devices -Part 10: Irritation 33.3 wt. % and Sensitization Tests; ISO 10993-10,1995. Kligman Sensitization Biological Testing of Medical and DentalNon-sensitizing at Materials and Devices - Part 10: Irritation 33.3 wt.% and Sensitization Tests; ISO 10993-10, 1995. Material MediatedBiological Evaluation of Medical Devices - Passed Pyrogen Assay Part 11:Tests for Systemic Toxicity; ISO 10993-11, 1993. Systemic InjectionBiological Evaluation of Medical Devices - Passed Part 11: Tests forSystemic Toxicity; ISO 10993-11, 1993.

C. Preparation of Nonwoven Films

1. Nonwoven Film Comprising Hydratable Keratin Fibers

Hydratable keratin fibers may be incorporated into a nonwoven film byadmixing with synthetic fibers which serve as a binder. Such a nonwovenfilm can be formed by mixing synthetic fibers made from, but not limitedto, α-olefins, acrylates, urethanes, acetates, nylons, esters, orcopolymers thereof with water-absorbent keratin fibers and heat pressingthe mixture into a film of desired thickness. The synthetic fibers willserve as a binder for the keratin fibers, while not completelyencapsulating them. This morphology provides mechanical integrity to thefilm, while allowing the keratin fibers to absorb water. The hydratedfibers can also release material which has been shown to be beneficialfor repairing damaged epithelial tissue.

Nonwoven films can be prepared by preparing nonwoven webs of a syntheticpolymer and then placing a layer of hydratable keratin fibers betweentwo layers of the nonwoven polymer-web. For example, a nonwoven film wasproduced by first preparing a nonwoven web measuring approximately onehalf inch thick by 24 inches wide using 9 denier, 38 mm lengthpolypropylene fibers. The web was made using a Rando-Webber, (FiberControls, Inc. Gastonia, N.C.) air laying machine operating at 2000 rpm,12 ft./minute let off speed with a feed rate of 4 ft./minute. A web ofapproximately 20 feet in length was coated over half of its length onone side with keratin fibers of approximately 2-5 mm in length. Thekeratin was spread on the web using a hand sifter. The uncoated sectionof the web was folded back over the coated section to form a laminate ofkeratin between two layers of polypropylene. The laminate was passedthrough a Sigma heated roller press (BF Perkins, Rochester N.Y.). Therolls were oil heated to 160° C. and a pressure of 350 pounds per linearinch was applied. The surface of the top roller was textured so as toimpress a texture in the finished nonwoven film. The laminate was fedthrough the rollers at approximately 4 ft./minute and the polypropylenesoftened and pressed such that a film of approximately 3 mm in thicknessresulted. This nonwoven film was bound together by the polypropylene,but retained some flexibility. The keratin fibers were at leastpartially exposed such that the film wetted easily and the keratinbecame gelatinous upon addition of water.

Nonwoven films can be made by other procedures. For example, if a moreopen nonwoven is desired, a laminate of keratin and synthetic fibers canbe prepared as described above, and this laminate processed by a throughair dryer. The through air dryer is capable of heating the laminate butdoes not apply pressure to the film. In this process, the syntheticfibers can be softened and bound together, thus providing a structuralmatrix for the keratin fibers. The result is a nonwoven web whichretains more of its original, open morphology. Also, films made withsynthetic fibers can sometimes be stiff. The example given aboveresulted in a film resembling burlap. If a softer film is desired,alternative fibers or blends of fibers may be used to produce thenonwoven web. A blend of cotton and polypropylene, for example, wouldprovide a softer, more pliable nonwoven film. Cotton fibers canconveniently be blended into the nonwoven web during the air laying orcarding process, prior to coating with keratin fibers. Other naturalfibers such as hemp may also be used.

These nonwoven films are produced from a loose, laminated precursor.However, the keratin fibers are exposed to the surface in the fmalproduct. Although the exemplified polymeric binder is hydrophobic, thenonwoven film wets easily and readily absorbs water. Once water isapplied to the film, the keratin fibers absorb it and swell, thusforming a hydrogel which is entrained in the unswollen binder. This typeof film is of utility as a wound dressing because of the capability ofabsorbing wound exudate and forming a hydrated, gelatinous cover overthe wound site. Such a dressing provides a closed, moist environment,conducive to wound healing. Due to the water absorbency, these nonwovenfilms also have utility as components of disposable diapers, femininehygiene products as well as any other application where a nontoxic filmwith biocompatibility and absorbency is desired and the healing ofdamaged skin or other epithelial tissue is deemed beneficial ornecessary. These films also have utility as implant materials for therepair of damaged hard or soft tissues, as cell scaffolds andtissue-engineering applications.

2. Nonwoven Film Comprising Hydratable Keratin Powder

Nonwoven films can also be prepared with hydratable keratin powder. Forexample, a nonwoven web measuring approximately one half inch thick by24 inches wide was prepared using 1.7 denier, 38 mm length Fortrel®polyester fibers supplied from Wellman, Inc. (Johnsonville, S.C.). Ablend of 20 wt. % low melt and 80 wt. % high melt fibers was first mixedby hand, then run through a Garnett fine opener, and finally carded.This was done prior to laying the web to provide a homogeneous sample.The web was made using a Rando-Webber (Fiber Controls, Inc, Gastonia,N.C.) air laying machine operating at 2000 rpm, 12 ft./minute let offspeed with a feed rate of 4 ft./minute.

The web was mechanically entangled using a hydrobonder from HoneycombSystems, Div. (Division of Valmet, Inc., Biddeford, Me.). This equipmentconsists of a screen conveyor and a manifold of high pressure waterjets.The web passes under the waterjet manifold and the force of the waterforces the fibers through the screen, thereby entangling them. Thedegree of entanglement can be controlled by the mesh size of the screenconveyor. The excess water was removed using a vacuum strippermanufactured by Evac Corporation, (Spartanburg, S.C.). This processreduced the web's thickness to approximately one eighth inch andresulted in a more tightly entangled web with more structural integritythan one produced using only the air laying technique.

Two rolls of web, 20 feet in length, were prepared using this processand used to make a laminate with hydratable keratin powder. The keratinpowder was less than 300 μm in size and was prepared as grindinghydratable keratin fibers. The laminate was prepared by conveying thetwo webs from separate spools and spraying the powder onto the bottomweb. Powder was sprayed using a GEMA™ powder sprayer with anelectrostatic spray gun (the electrostatic feature was not used). Thegun was operated at 2 psi with a flow of 4.5 m³/hour through thereservoir and a make-up flow of 1.5 m³/hour through the gun. Thenonwoven laminate was conveyed with a take-up winder operating at 32ft./minute. The use of a tighter web allowed small keratin particles(length of less than 1 mm) to be used without significant loss. This wasespecially important during the winding and unwinding operations priorto thermal bonding. The web could also be moistened slightly prior tospraying the keratin in order to promote adhesion.

The nonwoven laminate was passed through a Sigma heated roller press.The rolls were oil heated to 160° C. and a pressure of 200 to 215 poundsper linear inch was applied. The surface of the top roller was texturedso as to impress a texture in the finished nonwoven film. The laminatewas fed through the rollers at approximately 15 to 17 ft./minute. Thisprocedure resulted in a nonwoven film of approximately 3 mm inthickness. The surface of the film was smoother than the film describedpreviously and the use of polyester, rather than polypropylene, produceda softer, more pliable film. The keratin powder was at least partiallyexposed such that the film wetted easily and the keratin becamegelatinous upon addition of water.

D. Keratin Delivery Systems

Active agents can be incorporated into a hydratable keratin excipient toform a delivery system. By “active agent” is meant a compound, thedelivery of which is the object of the application of the preparationcomprising that compound and the keratin excipient of the presentinvention. Delivery of an active agent is generally desired because of abeneficial effect imparted by the agent upon delivery. Physical classesof active agents that can be incorporated into the keratin excipient ofthe present invention include, but are not limited to, compounds thatmay ion exchange with sulfonic acid groups, those compounds that mayotherwise be formulated as hydrochlorides, compounds that form anelectrostatic association with the keratin excipient, polar agents,polynucelotide agents, and polypeptide and peptide agents. Polypeptideagents include both recombinant and native polypeptides. Polar compoundsinclude, but are not limited to 4-acetaminophenol, aspirin andbeta-lactams. Compounds that may otherwise be formulated ashydrochlorides include, but are not limited to phenylpropanolamine andpseudoephedrine.

The active agent may be a pharmaceutical agent. By “pharmaceuticalagent” is meant any compound commonly referred to as a “drug,” orequivalents which include any physiologically or pharmacologicallyactive substance that produces a localized or systemic effect or effectsin animals. The term “animal” includes mammals, humans and primates suchas domestic, household, sport or farm animals such as sheep, goats,cattle, horses and pigs, laboratory animals such as mice, rats andguinea pigs, fishes, avians, reptiles and zoo animals Examples ofpharmaceutical agents that may be formulated as hydrochlorides, andexamples of pharmaceutical agents in general, are found in Remington:The Science and Practice of Pharmacy, (19th ed., ed. A. Gennaro) 1995,The Pharmacological Basis of Therapeutics, by Goodman and Gilman, 6thEd., 1980, published by the MacMillan Company, London and in The MerckIndex, 11th Edition, 1989, published by Merck & Co., Rahway, N.J.,herein incorporated by reference in their entirety. A non-exhaustivelist that exemplifies some of the classes and types of pharmaceuticalagents that may be used in the present invention is provided in Table 2.

TABLE 2 Analgesics aspirin, acetaminophen, morphine, oxymorphone,codeine, oxycodone Antianxiety Drugs buspirone, benzodiazepine,venlafaxine Antiarrhythmics flecainide, encainide, lidocaine, digoxin,beta-blockers, procainamide Antibacterials beta-lactams,aminoglycosides, macrolides, clindamycin, tetracylin, quinolones,sulfonamides, trimethoprim- sulfamethoxazole, sulfisoxaole,sulfasalazine, Antibiotics penicillins cephalosporins,amnioglycosidases, macrolides, fluroquniolones, chloamphenicol,rifampin, vancomycin Anticonvulsants phenytoin, ethosuximide, valproate,diazepam Antifungals amphotericin B,clotrimaozole, econazole,fluconazole, flucytosine, griseofulvin, haloprogrin, ketoconazole,itraconazole, miconazole, nystatin, tolfanate, undecylenic acid,terconazole, triacetin Antihistamines alkylamines, ethanolamines,ethylenediamines, piperazines, phenothiazines, piperidinesAnti-Inflammatories betamethasone dipropionate, clobetasol propianate,amcinonide, halcinonide, fluocinolone acetonide, betamethasone alerte,flubiprofen, ibuprofen, indomethacin, ketoprofen, mefenamic, naproxen,phenylbutazone, suldinac Antivirals acyclovir, amantidine,didanosine,inosiplex, intrathecal, ribavirin, ganciclovir, triflurdine Cytotoxicsprednisolone, azathioprine, cyclophosphamide, cyclosporine, tacrolimusCytokines inteferon alpha, interferon beta, colony stimulating factors(GM-CSF, M- CSF, G-CSF), interleukins 1 through 11, tumor necrosisfactor beta Growth Factors platelet-derived growth factor, epidermalgrowth factor, fibroblast growth factor, insulin-like growth factors,transformin growth factor beta Muscle Relaxants benzodiazepines,imidazopyridine, diphenhydramine, pyrilamine Sympathomimeticsphenylpropanolomine, phenylephrine, psudoephedrine Vitamins A, B₆, B₁₂,C, D, E, folacin, thaimin, riboflavin, niacin, pantothenic acid, biotin

The active agent may be a cosmetic agent. The term “cosmetic” used inrelation to a formulation or product means a formulation or product thatqualifies as a cosmetic under the Federal Food, Drug and Cosmetic Act,21 U.S.C. §321(i). By “cosmetic agent” is meant an agent that isincorporated in a cosmetic formulation or product and that agent isreported or believed to impart a beneficial effect upon application ofthe cosmetic. Cosmetic agents include, but are not limited to,anti-wrinkle agents, such as retinol and alpha-hydroxy acids,polypeptide and peptide agents derived from skin proteins (e.g.,including keratin, collagen and elastin), sunscreens, humectants,antioxidants, vitamins, tanning agents (both artificial and those thateffect melanogenesis), and whitening agents. Descriptions of cosmeticagents may be found in International Cosmetic Ingredient Dictionary andHandbook, Cosmetic Toiletries and Fragrance Association, 8^(th) ed.2000, and A Consumers Dictionary of Cosmetic Ingredients, Ruth Winter,Three Rivers Printers, 5^(th) ed, 1999, both herein incorporated byreference. The term “therapeutic agent” as used herein means bothpharmaceutical agents and cosmetic agents.

In some embodiments of the present invention, the keratin excipient maybe incorporated in a nonwoven fihn. In other embodiments of the presentinvention, the keratin excipient may be incorporated into a drugdelivery device. Examples of systems that may be adapted for transdermaluse with the compositions described herein described in U.S. Pat. Nos.4,816,252; 5,122,382; 5,198,223; 5,023,084; 4,906,169; 5,145,682;4,624,665; 4,687,481; 4,834,978; and 4,810,499 (all incorporated hereinby reference). Examples of systems that may be adapted for inhalation ofthe compositions described herein are described in U.S. Pat. Nos.5,884,620 and 5,960,792, both incorporated herein by reference.

The active agent delivery system of the present invention offersdistinct advantages over conventional drug dosage forms. As with mostdelivery systems, sustained or controlled release allows the level of anagent to be maintained at a more consistent concentration, therebyallowing larger doses to be administered on a less frequent basis. Inthe system described here, the chemical and material properties of thekeratin determine the properties of the dosage form. For example,loading can be varied by the availability of sulfonic acid bindingsites, which can in turn be controlled by the keratin oxidation process.Further, the disintegration and breakdown of the keratin can also becontrolled by the relative amount of disulfide crosslinks remainingafter the oxidation process. Disintegration and dissolution will effectthe release kinetics of the dosage form. When incorporated into anonwoven wound dressing, the bound drugs can be tailored to those mostbeneficial to wound healing such as, for example, antibiotics, biocides,pain medications and growth factors.

Following are examples of preparation, testing and formulation ofkeratin delivery systems:

1. Dissolution Testing

(a) Rotating Paddle Method

Dissolution tests are performed according to the standard United StatesPharmacopoeia (USP) rotating paddle method. In this method, the volumeof the dissolution medium is fixed and agitation is provided underdefined conditions by the stainless steel paddle. A 1 L volume ofpurified water or simulated gastric fluid (Lot. No. 76H9312,Sigma-Aldrich, St. Louis, Mo.) was used at 37° C.±0.5° C. Thetemperature of the dissolution medium was maintained at 37° C. byimmersing the dissolution flask in a water bath. A cover was used on thesystem to avoid water loss through evaporation. After the temperatureequilibrated, the samples were allowed to sink to the bottom of thevessel before rotation of the paddle. Rotation speed was kept constantat 50 rpm.

During the test, small volumes of sample were withdrawn at various timepoints, for example, every 20 minutes within the first hour (including a0 time point) and every hour after that for 8 hours. Samples were takenfrom a precise point in the dissolution flask, halfway between thesurface of the dissolution medium and the top of the rotating paddle,and not less than 10 mm from the wall of the vessel. The volumewithdrawn during sampling was replaced with an equal volume of media,pre-warmed to 37° C. High-performance liquid chromatography (HPLC) wasperformed on the samples and the amount released from the dosage formcalculated based on a calibration curve generated from solutions withknown concentrations of the compound of interest.

(b) Franz Diffusion Cell

An alternative dissolution test for membranous or film topicalformulations is by use of one-chambered Franz type diffusion cells(Franz, 1978). Franz cells may be obtained from Crown Glass (SomervilleN.J.). In these cells, one side of a membrane is in contact with anaqueous solution and the other side is open to the ambient atmosphere,unless placed within a controlled atmosphere. The keratin-containingfilm or membrane is mounted in the diffusion cell such that one side isin direct contact with the aqueous receptor solution (Lee et al., 1986).The aqueous solution is continuously stirred and kept at 32° C. by meansof a water jacket.

Samples are removed from the aqueous medium by means of the samplingport at appropriate times. For example, samples may be taken every 20minutes within the first hour (including a zero time point) and everyhour after that for 8 hours. An additional sample may be taken at 24hours. High-performance liquid chromatography (HPLC) or other suitableanalytical quantification is performed on the samples and the amountreleased from the dosage form is calculated based on a calibration curvegenerated from solutions of known concentrations.

2. Albumin Preparations

Samples containing albumin were made in tablet and capsule forms.Tablets were prepared by co-grinding a mixture of 80/20 keratinabsorbent powder/bovine albumin. The keratin absorbent powder wasprepared as described in Section VI. B supra, and the albumin waspurchased from Sigma Chemical Co., St. Louis, Mo. (Lot No. 69H1257,Fraction V, 96% purity). The solid mixture was further homogenized byshaking in a closed container. Samples of 500 mg of the powder mixturewere made into tablets using a pellet press at 30,000 psi (400 mgkeratin, 100 mg albumin). The dissolution profile of these tablets wasmeasured by the rotating paddle method.

Albumin capsules were prepared by dissolving 2 g of albumin in 200 g ofdeionized water. The albumin solution was added to 8 g of absorbentkeratin powder to form a hydrogel. The hydrogel was thoroughly mixed andpoured into petri dishes. The water was removed under vacuum at roomtemperature. After drying for approximately 36 hours, the resultingsolid was ground using a mortar and pestle. 400 mg of the groundkeratin/albumin powder (320 mg keratin/ 80 mg albumin) was placed intoeach of several two-part gelatin capsules (No. 0, Eli Lilly and Co.,Indianapolis, Ind.). The dissolution profile of these tablets wasmeasured using the rotating paddle method.

The results of the dissolution testing are summarized in the graph inFIG. 1. When the albumin was mechanically mixed with absorbent keratinin powder form (i.e. tablets), the disintegration and release werealmost immediate and complete in water. Release from the tablet ingastric fluid was also immediate, although the equilibrium amount ofalbumin was significantly lower, suggesting that some of the albuminremained bound to the keratin. This is presumably due to a pH effect oneither the keratin or the solubility or stability of the albumin ingastric fluid. An identical trend is evident in the data obtained fromcapsules.

Comparison of the tablet and capsule data reveals the effect of thedifferent preparation procedures that were employed. In the case of thetablets, simple mechanical mixing resulted in more material ultimatelybeing released, particularly in purified water, because mechanicalmixing does not result in a high degree of interaction at the molecularlevel. When intimate mixing was employed, as in the case of the gelatincapsules, much less material was released over an equivalent timeperiod. The intimate mixing was the result of first forming a gel froman albumin solution and keratin. In the hydrated state, albumin andkeratin molecules are more free to interact on a molecular level. Whilenot being bound to any theory, it is expected that intimate mixing wouldresult in more tightly bound albumin and consequently, less release thana system employing less intimate mixing. The unreleased material will beavailable however, and would be dissolved as the keratin degrades in abiological system.

3. Phenylpropanolamine (Norephedrine) Preparations

Samples that incorporated the vasoconstrictor norephedrine were preparedby binding the drug to oxidized keratin in an ion exchange step. Sixdifferent oxidized keratin samples were prepared by boiling 30 g each ofhuman hair in 500 mL of 2 w/v % H₂O₂ for 1, 2, 3, 4, 5 and 6 hours.Longer oxidation times results in the generation of more sulfonic acidresidues. More sulfonic acid residues results in increased bindingaffinity which in turn results in increased drug loading capabilitiesfor molecules that bind with sulfonic acid residues. Each of theoxidized keratin samples were ion exchanged with norephedrine by theproposed mechanism shown in FIG. 2. This was accomplished by dissolvinga measured amount of norephedrine into ethanol, adding 5 g of oxidizedkeratin and heating to reflux for 2 hours, followed by stirring at roomtemperature for 24 hours. Measured amounts of norephedrine weredetermined based on the amount of time the hair had been oxidized. Forexample, 5 g of hair oxidized for 4 hours would be exchanged withapproximately 5×10⁻³ moles of norephedrine. This calculation is based ondata generated for a pH 7 titration curve for H₂O₂ oxidized hair whichis shown in FIG. 3.

After binding the norephedrine in the ion exchange step, the solidkeratin was separated by filtration, dried, ground and pressed into 500mg tablets as described previously. The dissolution profile of thesetablets was measured using the rotating paddle method.

These data, displayed in FIG. 4, show a general trend toward higher drugloading at longer oxidation times, which is also suggested by thetitration curve shown in FIG. 3. As discussed previously, longeroxidation times results in increased sulfonic acid residues, which inturn increases drug loading for molecules like norephedrine. Inaddition, longer oxidation times resulted in more sustained release asdemonstrated by the 5 hour and 6 hour oxidation samples.

4. 4-Acetminophenol (Acetaminophen) Preparations

Samples that incorporated the antipyretic acetaminophen were prepared bybinding the drug to oxidized keratin in an ion exchange step. Sixdifferent oxidized keratin samples were prepared by boiling 30 g each ofhuman hair in 500 mL of 2 w/v % H₂O₂ for 1, 2, 3, 4, 5 and 6 hours. Thiswas done in order to generate samples with different amounts of sulfonicacid residues, hence, different binding affinities and presumably,different release characteristics. Compounds with weak ability to formamine salts, such as acetaminophen, are expected to form acid-baseinteractions with keratin molecules. Acetaminophen molecules alsocontain a phenolic functionality, which can participate in bindingthrough acid-base interactions. These binding mechanisms are similar tothe ion exchange process described for norephedrine, but result in aless tightly bound drug. Acid-base binding was accomplished bydissolving a measured amount of acetaminophen into ethanol, adding 5 gof oxidized keratin and heating to reflux for 2 hours, followed bystirring at room temperature for 24 hours. Measured amounts ofacetaminophen were estimated based on the amount of time the hair hadbeen oxidized. The pH 7 titration curve for H₂O₂ oxidized hair that isshown in FIG. 3 was used as a guide for the loading of keratin withacetaminophen.

After binding the acetaminophen in the ion exchange step, the solidkeratin was separated by filtration, dried, ground and pressed into 500mg tablets as described previously. The dissolution profile of thesetablets was measured using the rotating paddle method.

Evaluation of the loading of acetaminophen was accomplished bydisintegrating a single tablet into a known volume of water over a oneweek period at room temperature. In addition, that samples weresonicated for 90 minutes prior to sampling. A sample of the solution waswithdrawn and analyzed by HPLC.

The data are shown in FIG. 5 and FIG. 6. The curves in FIG. 5 do notsuggest a clear release trend relative to keratin oxidation time. Thismay be due to the fact that acetaminophen is a multifunctional moleculeand can potentially bind through more than one mechanism. However, thesemechanisms do not appear to result in tremendously strong interactions,or large numbers of interactions between acetaminophen and keratin. Thisis suggested by the overall low loading levels as shown in FIG. 6. Thedrug compound that does become bound, however, is released at a muchslower rate than norephedrine (i.e., more sustained release). This isparticularly true for the 4-hour oxidation sample.

E. Cell Scaffold and Tissue Engineering Applications

The use of implantable organ or tissue equivalents has been the subjectof considerable research and development. One approach is to attachisolated cells onto biocompatable scaffolds in vitro and then implantthe polymer-cell scaffold into recipients (Vacanti, 1988). Methods andexamples of the preparation of scaffolds and their use to implantdiverse cell types are disclosed in U.S. Pat. Nos. 5,041,138, 5,736,372,5,759,830, 5,770,193, 5,770,417, 5,964,807, 5,916,557 and 5,981,825,which are herein incorporated by reference. An alternative to the growthof cells on a scaffold in vitro for subsequent implantation, is the useof scaffolds to provide matrices for the infiltration and growth ofendogenous cells in vivo. Examples of such are disclosed in U.S. Pat.Nos. 4,772,287 and 4,904,260, which are herein incorporated byreference. Thus tissue engineering scaffolds can be used for tissuerepair, tissue reconstruction and wound healing, as disclosed forhyaluronan based scaffolds in U.S. Pat. No. 5,939,323, hereinincorporated by reference.

Various hydrogels have been used to form three-dimensional scaffoldssuitable for tissue engineering. These include hydrogels made from2-hydroxyethyl methacrylate (Plant et al., 1995; Santin et al., 1996),agarose (Bellamkonda et al., 1995, Dillon et al., 1998; Yu et al.,1999), gelatin (Kang et al., 1999), alginates (de Chalain et al., 1999;Rowley et al., 1999), and polysaccharide augmented glycosaminoglycan(Sechriest et al., 2000). In addition to the formation of a scaffold toguide growth, injection of cells in hydrogels have been shown to beretained at the delivery site and promote formation of the desiredregenerated tissue (Atala et al., 1993; Cao et al. 1998).

Keratins supply suitable substrata for the growth of cells. A keratinsheet has been disclosed to support the proliferation of humankeratinocytes, fibroblasts and microvascular endothelial cells (U.S.Pat. No. 5,932,552). In addition, it has been shown that akeratin-coated substrata was more adhesive to fibroblasts thancomparative collagen or glass substrata (Yamauchi et al., 1998). Akeratin implant material has been disclosed in U.S. Pat. No. 5,358,935,which is herein incorporated by reference. The keratin hydrogel of thepresent invention provides a suitable material for the construction ofcell scaffolds. For example, the absorbent keratin, which into ahydrogel upon the addition of water, can be used in the nonwoven filmembodiment of the present invention. The nonwoven film may beconstructed with synthetic polymer webs, the synthetic polymers of whichmay be resorbable. A number of synthetic polymers have been disclosed assuitable structural materials for tissue engineering scaffolds, such asin U.S. Pat. Nos. 5,399,665, 5,981,825, 5,981,825, and 6,022,828, whichare herein incorporated by reference. In other embodiments of thepresent invention, the absorbable keratin powder may be incorporatedinto synthetic polymer fibers or other three dimensional shapes.

The keratin hydrogel of the present invention may be used as a vehiclefor the injection of cells into a specific tissue location. For such avehicle, the absorbent keratin powder of the present invention would behydrated water or a medium suitable to maintain cell viability. Suchmedia are well known to those of ordinary skill in the art. Media can beadded to the absorbent keratin powder to tailor the viscosity forinjection. This viscosity will vary on the gauge size of the needleemployed and can be readily ascertained without undue experimentation byone of skill in the art. The injectable keratin hydrogel can also bedelivered to a support structure, which may be a permeable supportstructure, as disclosed in U.S. Pat. No. 6,027,744, hereby incorporatedby reference. The injectable keratin hydrogel can also be used as an invivo scaffold to fill soft tissue structural defects and replace tissue.The keratin hydrogel of the present invention is non-toxic andbiocompatible and may be used as a vehicle for various cell types,including, but not limited to keratinocytes, fibroblasts, chondrocytes,hepatocytes, splenocytes, osteoblasts, neurocytes and endothelial cells.

1. Hydrogel Stability

The stability of keratin hydrogels at body temperature were evaluatedfor injectable implant and tissue engineering applications. The testingmatrix employed varied keratin absorbent processing and formulationparameters. Processing parameters that were varied included oxidationtime (1-6 hr.), type of oxidant used (peracetic acid or hydrogenperoxide), and grinding time, which effected a change in the particlesize. Formulation parameters that were varied included solids content ofthe gel (1:8, 1:6 and 1:4 solids to liquid).

(a) Preparation of Hydrogels

The hydrogels were prepared as follows. Human hair obtained from abarber shop was washed with VersaClean™ detergent and dried by vacuumfiltration. The hair was oxidized by boiling in 2 w/v % solutions ofoxidant (either peracetic acid or hydrogen peroxide) at a solids toliquid ratio of ca. 1:17 for 1, 2, 3, 4, 5 and 6 hours. The oxidizedhair was rinsed with deionized water, filtered and dried. The pH of thehair was titrated to ca. 7 by exposure to a known amount of sodiumhydroxide. The number of moles of sodium needed to neutralize theoxidized hair was determined from titration curves for peracetic acidand hydrogen peroxide treated hair. Titration was affected by boilingthe hair in a solution of ethanol with sodium hydroxide for a 2 hourperiod. After cooling, the solution was continually stirred for anadditional 22 hours. The solid keratin was separated by filtration,rinsed with ethanol and dried under vacuum.

Grinding was performed in a 1 quart ball mill using 1 cm diameterceramic grinding media. Grinding times of 20, 40 and 60 minutes wereused so as to generate smaller keratin particles with longer grindingtimes. This resulted in the generation of 36 absorbent keratin powdersamples (2 oxidants×6 oxidation times×3 grinding times=36 samples). Eachpowder sample was then used to make hydrogels using pH 7.4 phosphatebuffered saline solution (lot no. 099H6118, [120 mmol/L NaCl, 2.7 mmol/LKCl and 10 mmol/L phosphate buffer], Sigma Diagnostics, St. Louis, Mo.63178). The weight ratio of solids to liquid was varied at 1:8, 1:6 and1:4, resulting in the generation of 108 samples (2 oxidants×6 oxidationtimes×3 grinding times×3 gels=108 samples). The gels were mixed in 4dram vials using a vortex mixer, capped, sealed with Parafilm™ andplaced in a heated oven at 37.5° C.±2° C. for in vitro stabilitytesting. Samples which did not form viscous gels were immediatelyremoved from the oven and discarded. Visual observations were conductedon a daily basis throughout the study. Gels that exhibited a decrease intheir original viscosity were removed from the study and deemed to nolonger be stable. These samples were retained frozen for later analysis.

(b) Stability and Viscosity Results

During the preparation of these gels, a variety of viscosities in theresulting hydrogels were observed. In general, gels with higher solidscontent were more viscous than gels with lower solids content(1:4>1:6>1:8). In addition, shorter grinding times (i.e. larger particlesize) also resulted in higher viscosities (20 min.>40 min.>60 min.).

The stability data collected are summarized in the attached FIGS. 7through 12. Formulations that were not stable for at least one day werecategorized as non gel-forming as indicated on the graphs. Most samplesthat were stable for greater than 80 days do not represent the terminusof the test, simply the cutoff point for data collection. To furtherexamine stability, the samples are further observed for greater than 80days until the samples are no longer stable or until some defined time.Samples which were stable at the last data collection time point areindicated with a data label on the graph.

In general, these data suggest that more stable gels were generated fromperacetic acid oxidized hair than from hydrogen peroxide oxidized hair(13 gels with stability greater than 80 days for peracetic acid versus 6for peroxide). Secondly, gels with higher solids content appear to bemore stable. Many more 1:4 gels were stable for long time periods thangels with a lower solids content. Lastly, these data do not show anydirect correlation between gel stability and particle size (i.e.grinding time).

In this study 19 gels exceeded 80 days of stability. To further testthese samples, they are subjected to elevated temperature testing.Primarily, they are gels made from hair that had been oxidized withperacetic acid for 3, 4 or 5 hours and formulated as 1:4 gels.

These data establish that keratin hydrogels can be processed to remainstable for time periods ranging from days to in excess of three months.At the time of degradation, the gel likely becomes bioresorbable. Thosegels that do not degrade are likely to remain biostable, thusdemonstrating a range of biodegradation with these gel formulations. Inaddition, the gels can be formulated to significantly vary viscosity,ranging from very fluid at low solids concentrations to highly viscousat high solids concentrations.

F. Applications of Hydratable Keratin

One use for the keratin powder and fiber is as a disposable diaperfiller material. Disposable diapers typically have an absorbent innerlayer which is often filled with a superabsorbent polymer and cellulosicmaterial, often chemically derived from wood pulp. In one application ofthe keratin material, a layer of the hydratable keratin is positioned ina disposable diaper near the skin but separated from the skin by apermeable layer. The hydratable keratin layer can serve to absorb urineand water from the wearer. In some embodiments, the hydratable keratinincludes a substantial fraction of soluble peptides having wound healingproperties, as discussed in co-pending U.S. patent application Ser. No.09/330,550, filed Jun. 11, 1999, entitled SOLUBLE KERATIN PEPTIDE, nowU.S. Pat. No. 6,270,791, herein incorporated by reference. Thewater-soluble peptides are believed to be entrained in the keratinstructure and able to leach out when water is applied. In use, thekeratin layer remains dry until soaked with urine, at which point thesoluble peptides can diffuse out of the keratin. The soluble peptidesdissolved in the liquid present can thus come in contact with the skin.The wound healing properties of the peptides are believed to bebeneficial in treating diaper rash.

In another use, the hydratable keratin powder or fiber can be used as aningredient in cosmetics. In one application, the keratin powder isadmixed with other cosmetic ingredients. The keratin power, when broughtinto contact with water from the other cosmetic ingredients or from theskin of the wearer, forms a hydrogel which forms a protective layer overthe skin and also retains moisture against the skin. The keratin powder,which has beneficial properties for skin, is thus held against the skin,moisturizing the skin. In some embodiments, the keratin powder includessoluble peptides which can diffuse out of the powder with application ofwater. The soluble peptides are believed to be non-immunogenic,mitogenic, and have beneficial skin healing properties. Cosmeticsincluding the hydratable keratin powder can aid in both moisturizing andhealing skin. Keratin powder can also be used as an absorbentreplacement to talc, the most popular cosmetic base, to which many areallergic. The keratin powder or fibrous material can be used to promotehealing of damaged skin. The keratin material can be applied to skinafflictions such as diaper rash, burns, sunburns, cuts, abrasions,punctures, sores, bed sores, ulcers, diabetic ulcers, irritated skin,surgical incisions, skin graft donor sites, and wrinkled skin. In onemethod, the keratin material is admixed with a carrier such as a cream,lotion, or gel.

Other applications of the keratin solid include using the keratin powderor fibers in feminine hygiene products, where the keratin can serve amoisture absorbing function. Yet another application is inanti-perspirants, where the keratin solid can absorb moisture. Stillanother application is in drug release applications, where the keratincan be used in powder, fiber, or film form to provide a moist, benignenvironment against the skin for drug release. An additional use as afood additive is contemplated, as some naturally-derived products, suchas gelatin, are already used in food products.

The keratin can also be used as the precursor to the formation of a gel,which can form a keratin hydrogel upon the addition of water to theabsorbent keratin solid. The keratin can be used to form an in situ gel.In the in situ application, the keratin powder can reside within anenvelope predisposed at a site and the water added into the envelopalready in position. The keratin can be stored in solid form, forexample as a fiber, powder, or some combination thereof, and water addedlater. Keeping the keratin in solid form allows for storage and latergel formation only when desired, as in an emergency medical fielddressing application. Requiring the keratin to pass through a solid stepalso serves to purify the resulting gel, as many impurities are removedin the intermediate processing steps.

The present invention can also be used to augment soft tissue. Keratinhydrogel precursor in powder form may be suspended in an injectablecarrier and injected subdermally. In one method, the keratin powder issuspended in saline and injected subdermally.

The resulting hydrogel has been observed to have viscoelasticproperties, favorable for use as an implant filler such as a breastimplant. The hydrogel has been observed to flow more readily whenmanipulated, which may prove beneficial to implant applications wherethe consistency of the implant is important.

The present invention can be used in a wound dressing. One use ofhydratable keratin in a wound dressing is wound exudate management. Thushydratable keratin may be used as a component of an absorbent wounddressing. In one embodiment, the hydratable keratin may be used in theform of a nonwoven film. In another embodiment, hydratable keratin maybe used as a woven composition. In a woven composition, fibers ofhydratable keratin may be woven with natural fibers or synthetic polymerfibers. In another embodiment, hydratable keratin is enclosed by awater-permeable material which, while allowing passage of wound exudateand blood, prevents passage of the hydratable keratin solid of thepresent invention. Wound dressings include, but are not limited to,adhesive bandages and tapes. Skin-contact adhesive bandages and medicaltapes are well known to those of skill in the art. Adhesive bandagestypically comprise an absorbent pad, a backing and a pressure sensitiveadhesive to maintain the dressing in place. In one aspect of the presentinvention, an absorbent wound dressing comprising hydratable keratin isan absorbent pad of an adhesive bandage. Components, configurations anddelivery systems for adhesive bandages are disclosed in U.S. Pat. Nos.6,018,092, 5,947,917, 5,633,070, and 5,503,908, which are hereinincorporated by reference. Upon contact with moisture, which may be inthe form of wound exudate or blood, the keratin fibers can form ahydrogel and leach water soluble peptides into the wound.

Numerous advantages of the invention covered by this document have beenset forth in the foregoing description. It will be understood, however,that this disclosure is, in many respects, only illustrative. Changesmay, be made in details, particularly in matters of reagents,concentrations, and step order, without exceeding the scope of theinvention. The invention's scope is, of course, defined in the languagein which the appended claims are expressed.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Atala et al., J. Urol. 150:745-47 (1993).

Bellamkonda et al., J. Biomed. Mater Res., 29:663-71 (1995).

Cao et al., Plast. Reconstr. Surg., 102:2293-98 (1998).

De Chalain et al., J. Biomed. Mater Res, 44:280-288 (1999).

Dillon et al., J. Biomatter. Sci. Polymer Ed., 9:1049-69 (1990).

Duranti et al., Dermatol. Sugery, 24:1317-25 (1998).

Franz, Curr. Probl. Dermatol., 7:309, 1978.

Kang et al., Biomaterials, 20:1339-44 (1999).

Lee et al., Drug Dev. Indust. Pharm., 12:349, 1986.

Manna et al., J. Eur. Acad. Dermatol. Venereol, 13:183-92 (1999).

Plant et al., Brain Res. 6:119-130 (1995).

Rowley et al., Biomaterials, 20:45-53 (1999)

Santin et al., Biomaterials, 17:1459-67 (1996).

Sechriest et al., J. Biomed. Matter Res., 49:534-41 (2000).

Vacanti, Archives of Surgery, 123: 545-549 (1988).

Yamauchi et al., J Biomater Sci Polym Ed, 9:259-70, 1998

Yu et al., Tissue Eng. 5:291-304 (1999).

What is claimed is:
 1. An cell scaffold comprising a nonwoven filmcomprising hydratable keratin containing sulfonate groups.
 2. Thescaffold of claim 1, wherein monovalent cations are ionically bound tosaid sulfonate groups.
 3. The scaffold of claim 2, wherein saidmonovalent cations are sodium or potassium.
 4. The scaffold of claim 1,wherein said hydratable keratin is obtained from hair, feathers, fur,skin, nails, horns or hooves.
 5. The scaffold of claim 1, wherein saidhydratable keratin is obtained from hair.
 6. The scaffold of claim 5,wherein said hair is human hair.
 7. The scaffold of claim 1, whereinsaid nonwoven film comprises a synthetic polymer.
 8. The scaffold ofclaim 7, wherein said synthetic polymer is a resorbable polymer.
 9. Thescaffold of claim 7, wherein said synthetic polymer is made from amaterial selected from the group consisting of α-olefins, acrylates,urethanes, acetates, nylons, esters, and copolymers thereof.
 10. Thescaffold of claim 7, wherein said nonwoven film is a laminate nonwovenfilm composition.
 11. The scaffold of claim 10, wherein said laminate isa tri-laminate comprising two outer layers of said synthetic polymer anda middle layer of said hydratable keratin.
 12. The scaffold of claim 11,wherein said middle layer of hydratable keratin is partially exposed byopenings in said two outer synthetic polymer layers.
 13. The scaffold ofclaim 1, wherein said hydratable keratin contains a therapeutic agent.14. The scaffold of claim 13, wherein said therapeutic agent is a watersoluble peptide.
 15. An implantable nonwoven scaffold made by theprocess: a. preparing a first nonwoven web layer comprising a syntheticpolymer; b. applying a hydratable keratin to one surface of said firstnonwoven web layer comprising said synthetic polymer; c. applying asecond nonwoven web layer comprising said synthetic polymer to saidhydratable keratin applied to said first nonwoven web layer to obtain atri-laminate composition comprising two outer layers of said first andsecond nonwoven webs comprising said synthetic polymer and a middlelayer of said hydratable keratin.
 16. The nonwoven scaffold of claim 15,wherein said hydratable keratin contains sulfonate groups.
 17. Thenonwoven scaffold of claim 16, wherein monovalent cations are ionicallybound to said sulfonate groups.
 18. The nonwoven scaffold of claim 17,wherein said monovalent cations are sodium or potassium.
 19. Thenonwoven scaffold of claim 15, wherein said hydratable keratin isobtained from hair, feathers, fur, skin, nails, horns or hooves.
 20. Thenonwoven scaffold of claim 15, wherein said hydratable keratin isobtained from hair.
 21. The nonwoven scaffold of claim 20, wherein saidhair is human hair.
 22. The nonwoven scaffold of claim 15, wherein saidmiddle layer of hydratable keratin is partially exposed by openings insaid two outer synthetic polymer layers.
 23. The nonwoven scaffold ofclaim 15, wherein said synthetic polymer is a resorbable polymer. 24.The nonwoven scaffold of claim 15, wherein said synthetic polymer ismade from a material selected from the group consisting of α-olefins,acrylates, urethanes, acetates, nylons, esters, and copolymers thereof.25. The nonwoven scaffold of claim 15, wherein said hydratable keratincontains a therapeutic agent.
 26. The scaffold of claim 25, wherein saidtherapeutic agent is a water soluble peptide.
 27. A method of supplyingan animal with a cell scaffold comprising implanting a cell scaffoldcomprising a nonwoven film comprising hydratable keratin containingsulfonate groups.
 28. The method of claim 27, wherein monovalent cationsare ionically bound to said sulfonate groups.
 29. The method of claim28, wherein said monovalent cations are sodium or potassium.
 30. Themethod of claim 27, wherein said hydratable keratin is obtained fromhair, feathers, fur, skin, nails, horns or hooves.
 31. The method ofclaim 27, wherein said hydratable keratin is obtained from hair.
 32. Themethod of claim 31, wherein said hair is human hair.
 33. The method ofclaim 27, wherein said nonwoven film comprises a synthetic polymer. 34.The method of claim 33, wherein said synthetic polymer is a resorbablepolymer.
 35. The method of claim 33, wherein said synthetic polymer ismade from a material selected from the group consisting of α-olefins,acrylates, urethanes, acetates, nylons, esters, and copolymers thereof.36. The method of claim 33, wherein said nonwoven film is a laminatenonwoven film composition.
 37. The method of claim 36, wherein saidlaminate is a tri-laminate comprising two outer layers of said syntheticpolymer and a middle layer of said hydratable keratin.
 38. The method ofclaim 37, wherein said middle layer of hydratable keratin is partiallyexposed by openings in said two outer synthetic polymer layers.
 39. Themethod of claim 27, wherein said hydratable keratin contains atherapeutic agent.
 40. The method of claim 39, wherein said therapeuticagent is a water soluble peptide.
 41. The method of claim 27, whereinsaid cell scaffold is seeded with a cell population prior toimplantation.
 42. The method of claim 41, wherein said cell populationis selected from group consisting of keratinocytes, fibroblasts,chondrocytes, hepatocytes, splenocytes, neurocytes, osteoblasts, andendothelial cells.
 43. A method for repairing tissue damage comprisingapplication of the cell scaffold of claim 1 to an area of damagedtissue.
 44. The method of claim 43, wherein said damaged tissue is softtissue.
 45. The method of claim 44, wherein said soft tissue is selectedfrom the group consisting of skin, mucosa and muscle.
 46. The method ofclaim 43, wherein said damaged tissue is hard tissue.
 47. The method ofclaim 46, wherein said hard tissue is bone or cartilage.